Muscles that move the retina augment compound-eye vision in Drosophila

The majority of animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all these species must resort to moving either their head or body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye1–3. Here we show that Drosophila use their retinal muscles to both smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. We show that when the retina moves, visual receptive fields shift accordingly and that even the smallest retinal saccades activate visual neurons. Using a new head-fixed behavioral paradigm we find that Drosophila perform binocular, vergence movements of their retinas—which could enhance depth perception—when crossing gaps and impairing retinal-motor-neuron physiology alters gap-crossing trajectories during free behavior. That flies evolved an ability to actuate their retinas argues that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlights a notable example of convergent evolution.

Eye movements serve many perceptual, cognitive, and social functions for primates and other species with single-lens eyes. Most animals, however, have compound eyes that attach rigidly to their heads. Drosophila melanogaster, for example, have compound eyes and this species serves as an important model for understanding the neural basis of vision. Might Drosophila, somehow, actively move their eyes even when their head is still? If so, what functions might such eye movements serve and how might their dynamics compare to eye-movement dynamics in primates and other vertebrates?
In the 1970s, it was discovered that houseflies have a muscle under each compound eye that attaches to the orbital ridge, a thin sheet of cuticle that surrounds the retina and part of the optic lobes 1,2 . This muscle seemed poised to shift the optical axes of the photoreceptors relative to the lenses of the compound eye 1,2,4 . "Clock spikes"-large, extracellularly recorded action potentials with regular inter-spike intervals evident in the vicinity of the optic lobes-were shown to reflect the activity of a motor neuron innervating this retinal muscle 2 . In 1991, a second muscle in houseflies was discovered, suggesting that the retina might move in two dimensions 3 . The rate of clock spikes is modulated spontaneously and also in response to external stimuli [4][5][6][7] . In preliminary reports, Franceschini and colleagues have argued that these spike-rate modulations correlate with movements of photoreceptors 3,8,9 . In Drosophila, recent work has focused on phototransduction-based movements of photoreceptors [10][11][12] and thus despite the initial housefly observations, the functions of muscular movements of the fly retina 13 remain unclear.

Two muscles move the Drosophila retina
We discovered that Drosophila, like houseflies, have two retinal muscles (Fig. 1a-c, Extended Data Movies 1,3). One muscle resembles the musculus orbito-tentoralis of large flies [1][2][3] . The muscle is attached via a long tendon to the tentorial bar posteriorly and inserts into the antero-medial rim of the orbital ridge. A second muscle, which resembles the musculus orbito-scapalis 3 of large flies, inserts more dorsally, into the frontal-medial rim of the orbital ridge. This second muscle originates at the edge of the antennal cup, to which it connects via a short tendon.
Because these muscles seem poised to move the Drosophila retina, we visualized the tips of the photoreceptors with a water-immersion objective positioned above the eye (Methods) 14 . We expressed the red-shifted channelrhodopsin, CsChrimson 15 , in the motor neurons innervating the retinal muscles. Via a pulse of red light, we optogenetically drove the retina to an extreme, tensed position (Fig. 1d, red) and then let it relax to an extreme, relaxed position by turning off the light (Fig. 1d, gray). We observed peak-to-peak displacements of the photoreceptors that spanned ~3 inter-photoreceptor spacings, which corresponds to ~15° in angular space (Fig. 1d).
Submerging the fly eye in water precludes many experiments. We thus decided to track the photoreceptor tips by measuring the position of the deep pseudopupil 16 -an enlarged, virtual image of photoreceptors that forms at the center of curvature of the compound eye, which can be visualized with an air lens (Fig. 1e). Specifically, when one places a point source of light abutting the fly, such that light exiting the compound-eye is collected by the air lens, the deep pseudopupil appears as seven large, bright dots, arranged in the shape of a single ommatidium's receptor array. We used 780 or 850 nm light-wavelengths to which the fly's natural opsins are insensitive 17 -to visualize the deep pseudopupil without blinding the fly. To verify that movements of the deep pseudopupil accurately reflect photoreceptor movements, we simultaneously tracked the deep pseudopupil (with an air lens) and photoreceptors at the top of the eye (with a water-immersion objective) and observed a tight correlation between these two signals (Fig. 1f, Extended Data Movie 4). We observed the same maximal angular excursion of ~15° in the deep pseudopupil during optogenetic activation of the retinal muscles as when tracking the positions of the photoreceptors directly (Fig. 1d-e).

Retinal movements shift receptive fields
Drosophila and other dipterans have neural superposition eyes, where each of the eight photoreceptors in a single ommatidium is in precise optical alignment to specific photoreceptors in neighboring ommatidia. Signals from all photoreceptors oriented along a common angle in visual space-independently of their ommatidium of origin-are ultimately combined in downstream neurons via a sophisticated neural wiring scheme 18 . If retinal muscles were to move the optical axes of photoreceptors in different ommatidia by different amounts, this would compromise the fidelity of neural superposition and thus impair the ability of visual neurons to signal effectively. To address this concern, we measured the visual responses of LC14 cells 19 (or dorsal cluster neurons 20 ) via wholecell patch-clamp recordings during optogenetically induced retinal movements. LC14 cells interconnect the two visual lobes 19,20 . We found that they have ~50° wide receptive fields along the frontal, vertical midline ( Fig. 2a-c). These cells respond strongly to moving bars or spots traversing their receptive fields (Extended Data Fig. 2).
Using a vertical bar that swept left or right in front of the fly, we measured the horizontal (yaw) position of LC14 receptive fields in the context of different retinal positions. To achieve two stable retinal positions, we either bilaterally activated retinal motoneurons optogenetically, or left them unactivated, as the bar swept. The example trace in Figure 2d shows consistent, depolarizing membrane voltage (Vm) responses to a bar moving across the receptive field, independently of whether the retina was optogenetically repositioned or not. Importantly, however, the trial-averaged Vm traces revealed that this LC14 cell responded to the bar at slightly different positions on the screen during optogenetic activation compared to control trials (Fig. 2e, red versus black). Because LC14 cells are insensitive to the direction of visual motion (Extended Data Fig. 2), we could combine trials in which the bar moved to the left and to the right to yield the best possible estimate of the shift in the cell's receptive field with optogenetic activation (Fig. 2f). All six LC14 cells that we recorded showed a receptive-field shift in the expected direction. Moreover, when we slid the population-averaged Vm curves with and without optogenetic activation by the mean pseudopupil (i.e. retinal image) displacement (5.7°), we observed a precise match of the Vm curves at the two retinal positions (Fig. 2g,h). The fact that retinal movements lead to precise shifts in the position of LC14 receptive fields, rather than degrading their visual responses more generally, argues that effective neural superposition is preserved at varied retinal positions.

Visually induced retinal movements
Many animals move their eyes to help stabilize gaze 21 . We wondered whether retinal movements might serve this function in flies. This would be a role akin to the optokinetic reflex in humans, where our eyes smoothly move in the direction of a rotating panoramic scene, which acts to minimize visual motion on our retinas. We tethered flies to tungsten pins with their heads rigidly glued to their thorax and placed them at the center of a panoramic LED display (Fig. 3a). We tracked the position of the pseudopupil of each eye independently. In some experiments, we had the flies perform tethered flight, in which case we simultaneously measured their wing-steering behavior (Methods).
When we rotated a panoramic squarewave grating around a quiescent, non-flying fly (Fig.  3b, left), we observed smooth, direction-selective, tracking movements of the retina in both eyes. Specifically, when the grating moved to the right, the retina moved smoothly to the left and vice versa. In flight, the same fly showed smooth tracking as well but with interspersed resetting movements in the other direction, i.e., counter-saccades (Fig. 3b, right), akin to nystagmus saccades in human optokinetic responses. Responses in flight were more variable, with varying frequency of nystagmus saccades on a trial-to-trial basis (compare left and right eye in flight). This fly's retinal movements were characteristic of the average movements in our population (Fig. 3c,d) (Extended Data Movies 5 and 6). Counter-saccades occur at unpredictable times and thus are not easily evident in the population-averaged responses (Extended Data Fig. 3). We note that in flight and in quiescence, we observed a tight correlation between the peak velocity of saccades and their amplitudes (Extended Data Fig. 4a,b), consistent with a power law relationship, reminiscent of the 'main sequence' in human saccades 22 .
The direction in which the fly retina moves in response to visual motion is opposite to the direction in which the lens of our own eye moves in response to the same visual stimulus, but both movements have the same slowing effect on visual motion. The key point is that when the human eye rotates, both the lens and retina move together whereas in Drosophila only the retina moves while the overlying lenses remain stationary. Because the lenses of the fly (and human) eye form inverted images, the fly retina, moving alone, has to move in a direction opposite to the direction of visual motion in order to slow down the movement of the image.
Unlike in humans, the optokinetic reflex in Drosophila can operate independently in each eye. When we presented a grating to the right eye only, the right retina tracked while the left retina remained stationary (Fig. 3e,f, Extended Data Movie 7) and vice versa (data not shown). Similarly, when we presented back-to-front motion to both eyes, this elicited front-to-back movements of both retinas (i.e. divergence) (Fig. 3g,h) and when we presented front-to-back visual motion to both eyes, both retinas moved back-to-front (convergence) (Fig. 3i,j).
The fact that Drosophila have two retinal muscles per eye ( Fig. 1) suggests that they can move their retinas in two dimensions. Indeed, flies also performed a vertical optokinetic response to up and down visual motion (Fig. 3k,l) with peak-to-peak magnitudes that were about 50% the size of horizontal movements.
The mean initial retinal speed in response to gratings moving horizontally at 15°/s was ~3°/s (Extended Data Fig. 5a-b) and thus too slow to fully cancel the visual motion experienced by the retina. This low gain is consistent with the values observed in mice 23 and goldfish 24 , but not with those observed in primates, where optokinetic gains can approach one 25,26 . It is unlikely that this incomplete cancellation of visual motion is due to muscular constraints, since the retina can move an order of magnitude faster during counter-saccades, with peak velocities exceeding 140°/s (Extended Data Fig. 4) and we also observed spontaneous saccades (described later) that exceeded 600°/s. In unrestrained animals, retinal movements likely work in synergy with head and body movements [27][28][29][30][31][32][33] to stabilize the visual image and thus they may not need to operate with a very high gain.
Recently it was argued that photocontraction 34 -photoreceptor movements resulting from the subcellular mechanics of rhabdomeric phototransduction-can induce Drosophila photoreceptors to physically move in vivo 10 . If the retinal movements we observed were due to photocontraction, they should persist in flies with intact photoreceptors but impaired downstream visual processing. We silenced the synaptic output of the major lamina visual neurons (L1-L4) 35,36 , which are monosynaptically downstream of photoreceptors, by expressing active tetanus toxin light chain in those cells 37 . Optokinetic responses to visual motion stimuli were entirely abolished after this manipulation (Extended Data Fig. 6), whereas control flies expressing an inactive form of the toxin showed robust retinal movements. These data alongside the bidirectionality of the optokinetic responsewhich requires the post-photoreceptor calculation of the direction of visual motion-and other results (Extended Data Fig. 7, Supplementary Text), strongly argue that the retinal movements we describe are not due to photocontraction.

Spontaneous retinal movements
Many animals move their eyes not only in response to external visual motion, but also seemingly spontaneously. We found that this is true in Drosophila as well (e.g., Extended Data Movie 8). Tracking the deep pseudopupil of a tethered, flying fly in the context of a stationary panoramic grating, we observed that the fly would keep its retina at a stable position for some time, move it rapidly to a new stable position, hold it there, then rapidly move it again, and so on (Fig. 4a, right). The sample fly in Figure 4a (right) performed fast retinal movements (Methods), or saccades, with amplitudes typically below 1°. A different tethered, flying fly performed larger saccades in the context of the same panoramic stimulus (Fig. 4b). In darkness, the pseudopupil of the sample fly in Figure 4a had an x-y position that drifted extensively, and the fly also exhibited larger saccades of up to ~5° (Fig. 4a, left). These data suggest that flying flies need structured visual input to keep their retinas stable.
We quantified the saccades flying flies performed in the context of a stationary grating and in darkness by detecting large saccades in one eye and plotting these alongside the concomitant retinal movement in the other eye (Fig. 4c). We observed significantly larger saccade amplitudes in flying flies in the context of a dark screen than with vertical gratings (means +/− standard deviation: 2.6° +/− 1.6 ° vs. 0.9° +/− 0.4) (Fig. 4c).

Small saccades activate visual neurons
Spontaneous retinal saccades often have amplitudes below 1°. Do such small movements of the retina have any impact on visual processing? We performed whole-cell patchclamp recordings from the horizontal and vertical system cells: motion-sensitive interneurons in the fly's lobula plate 38,39 . We observed direction-selective depolarizations and hyperpolarizations in response to tiny retinal movements (Extended Data Fig. 8), demonstrating that even the smallest retinal saccades are registered by the visual system (see Supplementary Text). Voltage responses to saccades were often small, however, in some flies we observed visual responses with magnitudes approaching the cell's full dynamic range as estimated by responses to drifting gratings (Extended Data Fig. 8b).

Retinal movements in gap crossing
We have shown that retinal movements impact visual-neuron physiology both by shifting receptive fields and by activating motion-sensitive neurons. How might retinal movements impact fly behavior (beyond improving image stability)?
Past work has argued that flies visually assess the length of a gap in deciding whether and how to cross it 40,41 . Specifically, if a gap is deemed crossable, Drosophila perform a set of leg-reaching movements to contact the other side, ultimately pulling themselves over; if a gap is uncrossable, flies are less likely to attempt to cross the gap at all 40 . Motion parallax has been suggested as one depth-estimating mechanism that flies might use in assessing depth during this task 40 . We wondered whether Drosophila might perform active retinal movements during gap crossing. We were particularly curious if flies perhaps performed active vergence movements as they crossed the gap; because flies have a binocular overlap zone of ~15° in the frontal visual field, such movements, in principle, could help flies to assess depth via either binocular triangulation or binocular-ruler mechanisms 2,42 (see Supplementary Text). Retinal movements during gap crossing could serve other roles as well.
Because we cannot as of yet measure retinal movements in freely moving flies, we developed a head-fixed gap crossing paradigm for Drosophila. Pin-tethered flies walked on a wheel that rotated along one axis 43 . The wheel included two, 2.5-mm wide gaps, 180° apart (Figure 5a). One gap had horizontal stripes on the walls and the other had vertical stripes (Methods); data associated with the two gaps have been combined because we noticed no consistent difference in the flies' behavior across them. We tracked the position of the wheel (Methods) as the flies walked with lights on for 15 min., in complete darkness for 15 min., and with the lights on again for 15 min. On average, flies crossed a gap in the forward direction every 51 s with the lights on and every 67 s in darkness, with considerable variability across individuals; that flies crossed gaps at a lower rate in darkness-alongside control experiments that showed no measurable optokinetic responses to a physically moving grating in darkness-argues that the flies could not see during the lights-off epoch (Extended Data Fig. 9). Whereas freely walking flies are unlikely to cross a gap that they cannot see, pin-tethered flies do so extensively, perhaps because their only navigational alternative, being rigidly tethered, is to walk backwards.
The example fly in Figure 5b consistently exhibited convergent pseudopupil movements (i.e., divergent optical axis movements across the two eyes) when crossing gaps (Fig. 5b, vertical grey lines) (Extended Data Movies 9-11). This observation held when we averaged retinal movements across all gap crossing events for this fly (Fig. 5c,e) and for a population of 23 flies (Fig. 5d,e). We observed similar vergence movements with the lights on and in darkness, albeit with more variability in darkness. That tethered flies made vergence retinal movements when crossing gaps in darkness argues that these movements reflect an active vision strategy rather than sensory responses to visual features of the gap. While moving the retina to improve visual perception is futile in darkness it is still attempted, likely as a reflex, much like a human would likely move their eyes in reflexive ways if forced to perform a visually guided task in darkness.
If retinal movements contribute to gap crossing, then impairing their dynamics might be expected to alter gap-crossing trajectories. Using two different split-Gal4 lines, we muted electrical signaling in retinal motor neurons by expressing in them a modified-mouse Kir2.1 ion channel; genetic-background matched controls expressed a mutated, non-conducting form of this channel 43 . We tracked freely walking flies crossing 3.5-mm long gaps. Gaps of this length are challenging but crossable in free behavior 40 (Fig. 5g,h). Control flies predominantly crossed the gap near the top, whereas experimental flies were more likely to walk down the near wall before crossing, yielding a statistically lower mean crossing height (y) for both experimental genotypes (y = −1.4 +/−0.1 mm, mean +− SEM, in silenced flies compared to −1.0 + −0.1 mm in control flies in one line, and y = −1.5 +/− 0.2 mm versus y = −1.0+/− 0.1mm in the second line; p<0.01 for both split-GAL4 lines, Welch Test) (Fig. 5i). A quantitative phenotype-i.e., 40-50% lower y-values-is consistent with the fact that expression of Kir2.1 yielded only a partial, ~33-35%, impairment to the magnitude of optokinetic retinal movements in both genotypes (Fig. 5j). Improved split-Gal4 lines (should they be possible to generate) that target the set of motor neurons more comprehensively and with higher transgene expression levels, should allow one to test the effect of more complete silencing of retinal movements on behavior in the future. Regardless, these data demonstrate that normal retinal-motor-system physiology is needed for flies to cross challenging gaps in a canonical fashion.

Discussion
Retinal optokinetic responses (Fig. 3) and spontaneous saccades (Fig. 4) in Drosophila conspicuously resemble human eye movements made in similar contexts. That the dynamics of an insect retina, actuated by two muscles, shows similarities to those of the vertebrate eye, actuated by six muscles of different origin, reveals a remarkable example of homoplasy in animal vision. Drosophila compound eyes have no equivalent of an area centralis or fovea, and many primate saccades are thought to be related to the act of foveation. Instead of foveation, spontaneous retinal saccades in flies could help to refresh the visual image in the face of receptor adaptation and we speculate that they may also improve the ability of flies to perceive fine spatial features by dynamically realigning the photoreceptors in relation to those features. Retinal movements, in principle, could also contribute to other functions in Drosophila such as spatial attention and visual object recognition, alongside providing a potential proxy measure for whether flies are awake, asleep, or experiencing varying levels of arousal.
We found the flies reliably perform vergence retinal movements when crossing gaps (Fig.  5). One function these movements might serve is to estimate the length of the gap via a binocular-ruler mechanism 42 (see Supplementary Text). Notably, LC14 neurons (Fig.  2) appear particularly well suited for implementing such a function 11,19,20,44 because they (1)  Calcofluor White. Soft tissue was removed proteolytically. It reveals the vesica piscis-shaped opening of the orbital ridge and shows strongly sclerotized parts in the frontal region, where the two muscles attach (red stars), as well as two discontinuities on the dorsal and ventral poles (arrows). One possibility is that these discontinuities decouple the front of the orbital ridge from the back, mechanically. This may allow muscles that pull the front of the retina to also move the rear by the same amount through internal cohesion within a stiff set of ommatidia, rather than through a force vector that dissipates over space from front to back. Alternatively, the inhomogeneities at the top and bottom of the orbital ridge could act as a fulcra or pivot points, leading to the rear part of the orbital ridge to move outward when the frontal part moves inward (towards the midline), which could aid coherent motion of the retina. We will test these models in biomechanical studies in the future. for full-field rightward motion, 1.6° and 0.7° for rightward motion in the right hemisphere and 2.7° vs 0.6° deg for bilateral front-to-back motion. All saccade-magnitude differences between flight and quiescence were highly significant (p<10 −10 , two-sided t-test). properly resolved (modified from Land 1997 52 ). Below the cut-off wavelength of λ L = 2·Δϕ, direction-selective motion responses of the visual system are predicted to invert due to spatial aliasing 52,53  The direction-selective responses to gratings argue that HS and VS cells respond to the visual motion induced on the retina by < 1° eye movements. The weak response to eye movements in darkness, or with a uniformly lit screen, is opposite in sign to that observed with a grating, which may represent an efference copy of the predicted motion signal arriving to HS/VS cells with each eye movement. This efference copy is potentially superseded by the actual, grating motion input with a high contrast grating, in the rightmost column.
Extended Data Figure 9. Evidence that flies are genuinely in the dark during the lights-off epoch of the gap crossing experiments.
(a) Flies walking on the gap-crossing wheel (Fig. 5) were presented with a grating printed on paper that was physically moved back and forth in front of the right eye with a motorized manipulator. A small slit in the printed grating allowed us to slide an InfiniStix lens through it, abutting the fly's right eye, to track the deep pseudopupil.   Fig. 2 and John Tuthill for suggesting the use of a specific split-GAL4 line to silence early visual neurons (Extended Data Fig. 5). We thank Haluk Lacin for sending us the split-GAL4 lines used in Figure 1, which also allowed us to ultimately make a more selective split-GAL4 line for retinal motor neurons. We thank Wyatt Korff for insights on the biomechanics of the orbital ridge. We thank Sachin Sethi for advocating for the head-fixed gap-crossing paradigm and Abigail Janke for performing optomotor flight experiments that did not make it into the final paper.

Data and code availability
The data shown in the main figures are available at https://doi.org/10.6084/ m9.figshare.c.6145572. All other data generated in this study are available from the corresponding authors upon request. Custom-written software used to track the fly retina in real time is available at https://github.com/MaimonLab/EyeTrackerForm. Additional code is available upon request from the corresponding authors.  subtracted membrane voltage for rightward bar motion in one fly. Red thin lines show trials with optogenetic activation (n = 9) and black lines trials without (n = 7), thick lines indicate the means. Right: As left, but for leftward bar motion (n = 9 for both conditions).   crossing-triggered averages revealed consistent convergent retinal movements at the moment of gap crossing. We plotted the sign-inverted product of the left-and right-eye retinal shifts (Methods) (top) as a metric that goes positive during coincident vergence movements. (d) Same as (c) but for a population of 23 flies. (e) Quantification of the time traces in (d). For the wheel position and the left-and right-eye retinal shifts, we calculated the mean baseline signal in a 3 s window, starting 5 s before gap crossing and we subtracted this value from the mean signal in a 3 s window starting 2 s after gap crossing. For the vergence metric we subtract the mean signal in a 1 s baseline window starting 2.5 s before gap crossing from the mean signal in a 1 s window surrounding the gap crossing event. All distributions are significantly different from zero (t-test, P<0.05, with a Bonferroni correction for 9 tests), except the vergence-measure distribution in darkness, which has a P value of 0.0083 that is just above the 0.0056 needed after the Bonferroni correction.